Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes. Bispecific antibodies may bind to two different epitopes of a single cell surface marker. Other such antibodies may bind a first cell surface marker and further bind a second cell surface marker on the same cell. A binding arm specific for a surface antigen of a target cell may also be combined with an arm which binds to a triggering molecule on a leukocyte such as a T cell receptor molecule (e.g., CD2 or CD3), or Fc receptors for IgG (FcγR), so as to focus cellular defense mechanisms to the target cell. Bispecific antibodies may also be used to localize cytotoxic agents to a target cell. These antibodies possess a target cell marker-binding arm and an arm which binds the cytotoxic agent (e.g., saporin, anti-interferon-α, vinca alkaloid, ricin A chain, methola-exate or radioactive isotope hapten). Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab′)).
Methods for making bispecific antibodies are known in the art. (See, for example, Millstein et al., Nature, 305:537-539 (1983); Traunecker et al., EMBO J., 10:3655-3659 (1991); Suresh et al., Methods in Enzymology, 121:210 (1986); Kostelny et al., J. Immunol., 148(5):1547-1553 (1992); Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993); Gruber et al., J. Immunol., 152:5368 (1994); U.S. Pat. Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,81; 95,731,168; 4,676,980; and 4,676,980, WO 94/04690; WO 91/00360; WO 92/200373; WO 93/17715; WO 92/08802; and EP 03089.).
MT103 is a recombinant, bispecific single-chain antibody or BiTE® molecule, which binds to CD19 on target cells and the CD3 complex on T effector cells. See Hoffmann P et. al., Int J Cancer. 2005 May 20; 115(1):98-104. It is a single polypeptide chain of 55 kDa with the variable binding domains of two murine monoclonal antibodies. By simultaneously binding to target and effector cells, MT103 triggers the T effector cell to form an immunological synapse, and to release perforin and granzymes which induce cell death of target cells. Target cells comprise human B cells of all development stages (with the exception of plasma cells) and malignant cells of most B cell-derived lymphoma and leukemia. Therefore, MT103 is currently in development for treatment of B-cell cancers.
Initial toxicology studies and clinical trials utilized a frozen liquid formulation which consisted of 1 μg/ml MT103 monomer in isotonic phosphate-buffered saline (pH 7.0-7.5) stabilized with 0.1% human serum albumin (HSA), sodium chloride, disodium monohydrogen phosphate, potassium dihydrogen phosphate and potassium chloride. A second lyophilized formulation was later developed to eliminate the use of HSA and enhanced the stability and recovery of MT103. This formulation consists of MT103 at 50 μg/mL in 25 mM citrate at pH 7.0, 500 mM lysine acetate, 15% trehalose dihydrate, 2.5% polyethylene glycol (PEG) 3350, 0.05% polysorbate 80. However, clinical use of the second lyophilized formulation never took place because the formulation presented significant interference in several essential analytical assays due to presence of the excipients lysine acetate and PEG 3350.
Therefore, further development was initiated to generate a formulation that 1) excludes the interfering excipients, 2) minimizes aggregate formation in the liquid bulks during storage, 3) maximizes the recovery of MT103 protein from the lyophilized product, 4) is suitable for intravenous, subcutaneous, or intramuscular administration, and 5) results in high bioavailability of the BiTE® molecules upon subcutaneous administration to a subject. Additional effort was put into optimization of the lyophilization cycle and robustness (boundary condition) studies.